Genomic Characterization of a Polyvalent Hydrocarbonoclastic Bacterium Pseudomonas Sp. Strain BUN14
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www.nature.com/scientificreports OPEN Genomic characterization of a polyvalent hydrocarbonoclastic bacterium Pseudomonas sp. strain BUN14 Mouna Mahjoubi1, Habibu Aliyu2, Mohamed Neifar1, Simone Cappello3, Habib Chouchane1, Yasmine Souissi1, Ahmed Salaheddine Masmoudi1, Don A. Cowan4 & Ameur Cherif1* Bioremediation ofers a viable alternative for the reduction of contaminants from the environment, particularly petroleum and its recalcitrant derivatives. In this study, the ability of a strain of Pseudomonas BUN14 to degrade crude oil, pristane and dioxin compounds, and to produce biosurfactants, was investigated. BUN14 is a halotolerant strain isolated from polluted sediment recovered from the refnery harbor on the Bizerte coast, north Tunisia and capable of producing surfactants. The strain BUN14 was assembled into 22 contigs of 4,898,053 bp with a mean GC content of 62.4%. Whole genome phylogeny and comparative genome analyses showed that strain BUN14 could be afliated with two validly described Pseudomonas Type Strains, P. kunmingensis DSM 25974T and P. chloritidismutans AW-1T. The current study, however, revealed that the two Type Strains are probably conspecifc and, given the priority of the latter, we proposed that P. kunmingensis DSM 25974 is a heteronym of P. chloritidismutans AW-1T. Using GC-FID analysis, we determined that BUN14 was able to use a range of hydrocarbons (crude oil, pristane, dibenzofuran, dibenzothiophene, naphthalene) as a sole carbon source. Genome analysis of BUN14 revealed the presence of a large repertoire of proteins (154) related to xenobiotic biodegradation and metabolism. Thus, 44 proteins were linked to the pathways for complete degradation of benzoate and naphthalene. The annotation of conserved functional domains led to the detection of putative genes encoding enzymes of the rhamnolipid biosynthesis pathway. Overall, the polyvalent hydrocarbon degradation capacity of BUN14 makes it a promising candidate for application in the bioremediation of polluted saline environments. Petroleum and dioxins compounds are omnipresent environmental pollutants that threaten the environment and human health due to their toxicological properties and their high resistance to degradation 1,2. Bioremediation is considered as one of the viable options for remediation of hydrocarbon-polluted environments 3,4. Numerous microorganisms, including bacteria, fungi, archaea and algae, have been investigated for their bioremediation potential3,4 with 79 bacterial genera reported to possess the capacity to degrade hydrocarbons 3. Members of the Gammaproteobacteria class have high hydrocarbon-degrading capacity, and this taxon includes the most obligate hydrocarbonoclastic genera: Alcanivorax, Talassolituus, Oleiphilus, Oleispira, Cycloclasticus and Mar- inobacter3,56. However, other non-obligate hydrocarbonoclastic members of the Gammaproteobacteria, such as the genus Pseudomonas, are well known for their hydrocarbon-degrading capacity 47. Te genetic basis and enzymatic mechanisms involved in the degradation of oil components, including alkanes and aromatic com- pounds, by Pseudomonas species have been reported in detail 8,9. Te key activating enzymes in the degradation of alkanes are alkane hydroxylases 10. Alkane monooxygenases (AlkB) and cytochrome P450s (CYP153) catalyze the hydroxylation of alkanes to alcohols, which are subsequently oxidized to fatty acids. Te fatty acid products are further metabolized by β-oxidation10. Te catabolism of aromatic compounds involves multiple enzymes which convert the aromatic substrates into Krebs cycle intermediates via ortho or meta-cleavage pathways. Te 1University of Manouba, ISBST, BVBGR-LR11ES31, Biotechpole SidiThabet, 2020 Ariana, Tunisia. 2Institute of Process Engineering in Life Science 2: Technical Biology, Karlsruhe Institute of Technology, Karlsruhe, Germany. 3Istituto per le Risorse Biologiche e le Biotecnologie Marine (IRBIM)-CNR of Messina, Sp. San Raineri, 86, 98122 Messina, Italy. 4Centre for Microbial Ecology and Genomics, University of Pretoria, Pretoria 0002, South Africa. *email: [email protected] Scientifc Reports | (2021) 11:8124 | https://doi.org/10.1038/s41598-021-87487-2 1 Vol.:(0123456789) www.nature.com/scientificreports/ l3-ketoadipate pathway (ortho-cleavage pathway) is the key route for the catabolism of a wide variety of aromatic compounds through the protocatechuate branch (pea genes) and the catechol branch (cat genes). Te phylogeny of the genus Pseudomonas is complex, with some 219 validly published and correctly named species11. However, the current phylogeny is subject to considerable debate12 and many of the named species may be considered as conspecifc. For example, Pseudomonas kunmingensis HL22-2T and P. chloritidismutans AW-1T which are closely related to P. stutzeri may not deserve separate species status 13,14. To date, the genome sequences of eighteen strains afliated to P. kunmingensis and one afliated to P. chl or- itidismutans strain are available via the NCBI database 15. While the genes and pathways associated with n-alkane degradation have been reported for P. chloritidismutans16,17, similar information for Pseudomonas kunmingensis is not available. Pseudomonas sp. BUN14, isolated from hydrocarbon-polluted sediments from the Bizerte coast refnery harbour, north Tunisia, exhibits the capacity for hydrocarbon degradation. In this study, we combined genomic analysis and degradation experiments to demonstrate that Pseudomonas strain BUN14 has potential for applica- tion in the bioremediation of hydrocarbon contaminated marine environments. Results and discussion Isolation, phylogenetic assignment, and characterization of hydrocarbonoclastic bacterial strain BUN14. Strain BUN14 was isolated on ONR7a mineral medium supplemented with 1% crude oil as the sole carbon source. Te comparison of the strain BUN14 16S rRNA gene sequence with those of validly described strains in the EzBioCloud18,19 revealed that strain BUN14 showed highest similarity (99.23%) to that of P. kunmingensis HL22-2T Phylogenetic analysis based on the 16S rRNA gene of Pseudomonas species placed P. kunmingensis HL22-2T as the closest relative of BUN14 (Fig. S1). Growth of strain BUN14 on plates containing TSA (Trypticase soy agar) medium showed that the bacterium formed circular, opaque yellow colonies. Cells were rod-shaped and approximately 0.6 ± 0.1 μm in diameter and 1.8–2.0 μm in length (Fig. S2). Optimization of surfactant activities. When grown on mineral medium ONR7a supplemented with crude oil and vegetable oil as sole carbon sources, BUN14 strain produced biosurfactants, as indicated by the oil spreading, drop collapse and emulsifcation tests (Fig. S2). Use of the Cetyl Tri Ammonium Bromide (CTAB)- Methylene blue agar method indicated that BUN14 produced anionic biosurfactant (Fig. S2). Optimal condi- tions for growth and biosurfactant production were assessed using response surface methodology (RSM) experi- ments. Te analysis of variance for the ftted mathematical models shows that the regression sum of squares was statistically signifcant (P < 0.01) (Table S5). Response surface plots, showing optimal biosurfactant production conditions, are shown in Fig. 1. Statistical analyses of central composite design (CCD) experiments demon- strated that the percentage of oil substrate, NaCl concentration, inoculum size and incubation time all afected the biosurfactant production. Based on desirability function, the optimal BUN14 growth (OD 600nm, 0.54), bio- surfactant production (OD 625nm, 2.58) and activities (emulsion index E24, 40.09% and oil-displacement ODA, 20.40 cm2), were obtained afer approx. 7 days of cultivation crude oil, 2.50% NaCl concentration inoculum size source (Fig. S3). Accordingly, as for previous studies20,21 substrate, NaCl concentration, inoculum size and incu- bation time signifcantly afected biosurfactant production (0.01 < p < 0.05). Te basic structure of the isolated biosurfactant was evaluated by Fourier Transform InfraRed (FT-IR) spec- trometry and compared to a reference Pseudomonas aeruginosa (Fig. 2). Te peaks appearing at 3278.56 (Fig. 2a) and 3270.42 cm−1 (Fig. 2b) denoted the presence of –OH stretching (free hydroxyl groups of rhamnose rings) of hydroxyl group. Te adsorption peaks at 2924.64 (Fig. 2b) and 3000.0 cm−1 (Fig. 2a) indicated the presence of terminal methyl group of aliphatic stretching bands (CH2, CH3). Te absorption peaks at 1097.35 (Fig. 2a) and 1049.1 cm−1 (Fig. 2b) confrmed the presence of C–O–C vibrations (rhamnose ring). Te area between 1495.92 and 1150.02 cm−1 (Fig. 2a) represented C–H and OH deformation vibrations typical for carbohydrates, as in the rhamnose units of the biosurfactant. Te apparent similarity of the main functional groups determined by FTIR spectrometry of the commercial rhamnolipid (R90) from Pseudomonas aeruginosa (AGAE Technologies, Corval- lis, OR, USA) and the isolated biomolecule from strain BUN14 (Fig. 2), suggested that the BUN14 biosurfactant product was composed on rhamnose rings with long hydrocarbon chains. Members of the genus Pseudomonas are known for biosynthesis of rhamnolipids with biosurfactant properties20,22. Hydrocarbon degradation. Strain BUN14 could grow on and utilize various hydrocarbons, including pyrene, naphthalene, phenanthrene, carbazole, dibenzofuran, dibenzothiohene, biphenyl, pristane, fuoran- thene, crude oil, octadecane and tetradecane as sole carbon and energy sources (Fig. S4a–c). Tese fndings are in agreement with previous reports demonstrating that